Despite extensive research on the Hb molecule, the mechanism by which heme and globin subunits coordinately assemble and how misfolded and unstable unassembled globin chains are removed from erythrocytes are not known. In addition, the basic mechanism by which Hb F inhibits polymerization and ameliorates the clinical course of SCD is not completely understood. Elucidating such mechanisms can contribute to the development of strategies for gene therapy in the treatment of diseases of altered globin chains or those associated with decreased globin synthesis. In this proposal we aim (1) gamma-chain assembly with a chains to form functional human fetal Hb, (2) Ubiquitin-mediated degradation of excess non-alpha globin chains in vivo, and (3) Engineered Hb F variants having low oxygen affinity and inhibitory properties on Rb S polymerization. The long-range goal is to identify and design optimal Rb F variants for use in gene therapy of sickle cell disease (SCD) andthalassemia. In Specific Aims (1) we will test two related hypotheses; (i) Folded alpha-globin chains assemble with intermediately folded nascent gamma-chains prior to or soon after the release from polyribosomes. (ii) The amino acids at G-10, 14 and 18, which have been shown by x-ray crystallographic analysis to be at the alpha1gamma1 interaction sites on the G helix, are critical for assembly of alpha- and gamma-globin chains in vivo as well as in vitro. In Specific Aim (2), we hypothesize that purified non-alpha chain tetramers, like Hb hetero-tetramers, are not substrates for ubiquitination since Beta4 and gamma4 structures are very similar to the alpha2Beta2 heterotetramer structure. Using a rabbit reticulocyte cell free system, we will measure degradation of non-alpha chain in the absence of a chain during translation in the presence of ubiquitin. In specific Aim (3), we will continue to investigate the inhibitory mechanism of Hb S polymerization by Hb F. We hypothesize that Hb F variants (e.g., Hb F gamma 73 His, Rb F gamma 6Val & 73 His) can be engineered that have inhibitory properties exceeding those of Hb F and we will seek such variants. We will also continue to seek Rb F variants with lower oxygen affinity than Hb S through not only enhancement of 2,3-BPG interaction but also amino acid substitution at the alyl interaction sites on the G helix. Because of their lower oxygen affinity, these hemoglobin variants in addition to having anti-nucleation properties would effectively inhibit sickling at lower levels than would native Rb F, such as about 10 percent vs. 20 percent. The understanding of the assembly of gamma and alpha chain and the mechanism of degradation of excess globin will provide a basis for determining the most appropriate gamma chain mutant for gene therapy, which should be one that can be introduced by viral vectors at significantly lower levels than native Rb F. Furthermore, these studies will be of general interest to researchers who study protein biosynthesis and will help identify why some mutant globin chains are incorporated into hemoglobin more or less efficiently than wild type chains as well as how separately translated alpha and non-alpha chain are quality controlled during hemoglobin formation to preserve functional erythrocytes.